Dynamics of detachment of Escherichia coli from plasma-mediated coatings under shear flow

A series of plasma-mediated coatings, containing silver nanoparticles embedded in an organosilicon or silica-like matrix, were deposited onto stainless steel and chemically characterized. Their anti-adhesive properties were evaluated in vitro towards Escherichia coli by performing shear-flow induced detachment experiments. Increasing the wall shear stress facilitated E. coli cell detachment, irrespective of the coating characteristics. When nanosilver was incorporated, cell detachment was lower, probably due to the affinity of the embedded silver for biological components of the cell wall. The presence of methyl groups in the matrix network could also promote enhanced hydrophobic interactions. Within the population fraction remaining attached to the coating under increasing shear flow, different association phenotypes were observed, viz. progressively lying flat, moving laterally, remaining tethered, or rotating by a single anchoring point, until alignment with the flow direction. This re-orientation phenotype and its relation with detachment were dependent of the coating. The effects of such heterogeneities should be more deeply explored.     

Transglutaminase stabilizes melanoma adhesion under laminar flow

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-crosslinkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (≥ 32.5 dynes/cm²). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm²). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked¹²⁵I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Shear stress increases the residence time of adhesion of Pseudomonas aeruginosa

Although ubiquitous, the processes by which bacteria colonize surfaces remain poorly understood. Here we report results for the influence of the wall shear stress on the early-stage adhesion of Pseudomonas aeruginosa PA14 on glass and polydimethylsiloxane surfaces. We use image analysis to measure the residence time of each adhering bacterium under flow. Our main finding is that, on either surface, the characteristic residence time of bacteria increases approximately linearly as the shear stress increases (∼0–3.5 Pa). To investigate this phenomenon, we used mutant strains defective in surface organelles (type I pili, type IV pili, or the flagellum) or extracellular matrix production. Our results show that, although these bacterial surface features influence the frequency of adhesion events and the early-stage detachment probability, none of them is responsible for the trend in the shear-enhanced adhesion time. These observations bring what we believe are new insights into the mechanism of bacterial attachment in shear flows, and suggest a role for other intrinsic features of the cell surface, or a dynamic cell response to shear stress.     

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m^?2 for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m^?2 and > a two-fold reduction in the storage modulus (G′). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.     

Microfluidic accumulation assay probes attachment of biofilm forming diatom cells

Testing of fouling release (FR) technologies is of great relevance for discovery of the next generation of protective marine coatings. In this paper, an accumulation assay to test diatom interaction under laminar flow with the model organism Navicula perminuta is introduced. Using time lapse microscopy with large area sampling allows determination of the accumulation kinetics of the diatom on three model surfaces with different surface properties at different wall shear stresses. The hydrodynamic conditions within the flow cell are described and a suitable shear stress range to perform accumulation experiments is identified at which statistically significant discrimination of surfaces is possible. The observed trends compare well to published adhesion preferences of N. perminuta. Also, previously determined trends of critical wall shear stresses required for cell removal from the same set of functionalized interfaces shows consistent trends. Initial attachment mediated by extracellular polymeric substances (EPS) present outside the diatoms leads to the conclusion that the FR potential of the tested coating candidates can be deducted from dynamic accumulation experiments under well-defined hydrodynamic conditions. As well as testing new coating candidates for their FR properties, monitoring of the adhesion process under flow provides additional information on the mechanism and geometry of attachment and the population kinetics.     

Transglutaminase stabilizes melanoma adhesion under laminar flow.

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Influence of Fluid Shear and Microbubbles on Bacterial Detachment from a Surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s⁻¹) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

Effects of glucanase treatment on the structure and mechanical properties of cell walls in the giant‐celled xanthophycean alga Vaucheria frigida

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.     

Dynamic Mechanisms of the Bactericidal Action of an Al2O3-TiO2-Ag Granular Material on an Escherichia coli Strain

The bactericidal activity of an Al₂O₃-TiO₂-Ag granular material against an Escherichia coli strain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics on Escherichia coli using different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al₂O₃-TiO₂-Ag material, 98% of E. coli isolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeable E. coli cells and 1% of intact cells (10⁵ genomic units · ml⁻¹) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced.     

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.     

Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress

An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid-generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time-lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm², respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell-substrate contacts. © 1994 wiley-Liss, Inc.     

Influence of flow rate variation on the development of Escherichia coli biofilms

This work investigates the effect of flow rate variation on mass transfer and on the development of Escherichia coli biofilms on a flow cell reactor under turbulent flow conditions. Computational fluid dynamics (CFD) was used to assess the applicability of this reactor for the simulation of industrial and biomedical biofilms and the numerical results were validated by streak photography. Two flow rates of 374 and 242 L h^?1 (corresponding to Reynolds numbers of 6,720 and 4,350) were tested and wall shear stresses between 0.183 and 0.511 Pa were predicted in the flow cell reactor. External mass transfer coefficients of 1.38 × 10^?5 and 9.64 × 10^?6 m s^?1 were obtained for the higher and lower flow rates, respectively. Biofilm formation was favored at the lowest flow rate because shear stress effects were more important than mass transfer limitations. This flow cell reactor generates wall shear stresses that are similar to those found in some industrial and biomedical settings, thus it is likely that the results obtained on this work can be used in the development of biofilm control strategies in both scenarios.     

Run and Tumble Chemotaxis in a Shear Flow: The Effect of Temporal Comparisons,Persistence, Rotational Diffusion,and Cell Shape

Escherichia coli is a motile bacterium that moves up a chemoattractant gradient by performing a biased random walk composed of alternating runs and tumbles. This paper presents calculations of the chemotactic drift velocity v _d (the mean velocity up the chemoattractant gradient) of an E. coli cell performing chemotaxis in a uniform, steady shear flow, with a weak chemoattractant gradient at right angles to the flow. Extending earlier models, a combined analytic and numerical approach is used to assess the effect of several complications, namely (i) a cell cannot detect a chemoattractant gradient directly but rather makes temporal comparisons of chemoattractant concentration, (ii) the tumbles exhibit persistence of direction, meaning that the swimming directions before and after a tumble are correlated, (iii) the cell suffers random re-orientations due to rotational Brownian motion, and (iv) the non-spherical shape of the cell affects the way that it is rotated by the shear flow. These complications influence the dependence of v _d on the shear rate γ. When they are all included, it is found that (a) shear disrupts chemotaxis and shear rates beyond γ≈2 s⁻¹ render chemotaxis ineffective, (b) in terms of maximizing drift velocity, persistence of direction is advantageous in a quiescent fluid but disadvantageous in a shear flow, and (c) a more elongated body shape is advantageous in performing chemotaxis in a shear flow. J.T. Locsei is supported by an Oliver Gatty Studentship from the University of Cambridge.     

In Vitro Evaluation of the Efficacy of a Silver-Coated Catheter

Bacteria commonly associated with nosocomial urinary tract infections were examined in vitro for their relative adherence to latex, 100% silicone-, hydrogel-coated latex-, and hydrogel/silver-coated latex urinary catheters. Degrees of adherence within 2 h were determined with cells radiolabeled with leucine. Adherence was greatest and equivalent on silicone and latex catheters. Adherence of four strains of Escherichia coli to the hydrogel/silver-coated catheter was decreased by 50% to 99% in comparison with the other catheters. Repeat testing with strains of E. coli and Pseudomonas aeruginosa with over 50 catheters demonstrated a consistency in the inhibition. The hydrophilic coating of the catheter appeared to be primary in the decreased adherence phenomenon followed by a secondary biocidal effect of the silver ion. Received: 2 December 1995 / Accepted: 3 January 1996     

Bactericidal silver ion delivery into hydrophobic coatings with surfactants

A much studied oil-soluble surfactant, bis[2-ethylhexyl]sulfosuccinate, sodium salt, was ion exchanged into the silver ion form and dissolved into microemulsions of immiscible polyurethane step monomers. Coating and curing of these microemulsions produced polyurethane coatings that exhibit bactericidal activity against representative Gram negative bacteria. After 24 h exposure, 0.006–0.012% weight Ag relative to coating weight (0.0013–0.0025 μmol Ag/cm²) results in the three-log reduction in Escherichia coli. A slightly higher level of 0.031% weight Ag relative to coating weight (0.006 μmol Ag/cm²) killed all of the E. coli after 12 h exposure. Similar results were obtained for Pseudomonas aeruginosa. Since the double-tail surfactant anion promotes reverse micelle formation in many different kinds of oils and solvents, it appears an excellent vector for incorporating low and effective amounts of silver ion into many industrial, hospital, and household coating formulations.     

Elevated Shear Stress Protects Escherichia coli Cells Adhering to Surfaces via Catch Bonds from Detachment by Soluble Inhibitors

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of Escherichia coli. Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.     

Comparison of ultrafiltration systems for concentration of biologicals

Ultrafiltration has been used with increasing frequency in recent years in biological laboratories for concentration, separation or purification of biological material. No data have been available on the comparison of the characteristics of Ultrafiltration systems currently in use. This study compares the filtration characteristics of four systems using commercially available membranes on suspensions and solutions: suspension of protein micelles (casein), cell debris (E. coli) and catalase solution. None of the four systems considered is found to be generally superior for all the suspensions and solutions. Vibration systems were most effective when relatively large particles were involved, while laminar flow recycling systems with high wall shear rates were best for dilute suspensions and proteins in solution. It was found that shearing inactivates enzymes in both recycle and vibration systems. It was also observed that vibration actually reduces flux in dilute solutions.     

Specific adhesion of glycophorin liposomes to a lectin surface in shear flow.

The adhesion of cells to other cells or to surfaces by receptor-ligand binding in a shear field is an important aspect of many different biological processes and various cell separation techniques. The purpose of this study was to observe the adhesion of model cells with receptor molecules embedded in their surfaces to a ligand-coated surface under well-defined flow conditions in a parallel plate flow chamber. Liposomes containing glycophorin were used as the model cells to permit a variation in the adhesion parameters and then to observe the effect on adhesion. A mathematical model for cell sedimentation was created to predict the deposition time and the velocity preceding adhesion for the selection of experimental operating conditions and the methods useful for data analysis. The likelihood of cell attachment was represented by a quantity called the sticking probability which was defined as the inverse of the number of times a liposome made contact with the surface before attachment occurred. The sticking probability decreased as the cell receptor concentration was lowered from approximately 10(4) to 10(2) receptors per 4-microns diam liposome and as the shear rate increased from 5 to 22 s-1. The effect of the wall shear rate and particle diameter on detachment of liposomes from a surface was also observed.     

Effect of Different Concentrations of Ortho-phthalaldehyde on Biofilms Formed by Pseudomonas fluorescens Under Different Flow Conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

Morphology and ultrastructure of Interfilum and Klebsormidium (Klebsormidiales,Streptophyta) with special reference to cell division and thallus formation

Representatives of the closely related genera, Interfilum and Klebsormidium, are characterized by unicells, dyads or packets in Interfilum and contrasting uniseriate filaments in Klebsormidium. According to the literature, these distinct thallus forms originate by different types of cell division, sporulation (cytogony) versus vegetative cell division (cytotomy), but investigations of their morphology and ultrastructure show a high degree of similarity. Cell walls of both genera are characterized by triangular spaces between cell walls of neighbouring cells and the parental wall or central space among the walls of a cell packet, exfoliations and projections of the parental wall and cap-like and H-like fragments of the cell wall. In both genera, each cell has its individual cell wall and it also has part of the common parental wall or its remnants. Therefore, vegetative cells of Interfilum and Klebsormidium probably divide by the same type of cell division (sporulation-like). Various strains representing different species of the two genera are characterized by differences in cell wall ultrastructure, particularly the level of preservation, rupture or gelatinization of the parental wall surrounding the daughter cells. The differing morphologies of representatives of various lineages result from features of the parental wall during cell separation and detachment. Cell division in three planes (usual in Interfilum and a rare event in Klebsormidium) takes place in spherical or short cylindrical cells, with the chloroplast positioned perpendicularly or obliquely to the filament (dyad) axis. The morphological differences are mainly a consequence of differing fates of the parental wall after cell division and detachment. The development of different morphologies within the two genera mostly depends on characters such as the shape of cells, texture of cell walls, mechanical interactions between cells and the influence of environmental conditions.